LincRNA-p21抑制食管癌细胞增殖的机制

Mechanism of Linc RNA-p21 on inhibiting proliferation of esophageal cancer cells

[目的]探讨基因间的长链非编码RNA(long intergenic non-coding RNA,LincRNA)-p21抑制食管癌细胞增殖的调控及机制。[方法]应用实时定量逆转录-聚合酶链反应(RT-qPCR)技术,检测LincRNA-p21在2株食管癌细胞(EC109和EC9706)与1株永生化食管上皮细胞(Het-1A)以及64例食管癌组织与癌旁组织中的表达情况。携带LincRNA-p21基因全长的慢病毒成功转染食管癌EC109后,通过流式细胞术检测食管癌细胞EC109的周期分布,Ed U染色法分析EC109的增殖情况。同时,采用RT-qPCR和Western blot技术分别检测LincRNA-p21下游基因p21的mRNA水平和蛋白表达水平,以及cyclin D蛋白水平。[结果]EC109和EC9706中LincRNA-p21水平分别为Het-1A的18%和32%(P<0.05);p21的mRNA水平分别为Het-1A的42%和48%。以EC109为靶细胞进行慢病毒转染后,LincRNA-p21相对表达量增加约14 860倍(P<0.05),p21的mRNA上调约1.83倍(P<0.05)。细胞周期分布结果显示,LincRNA-p21转染组其G1期细胞增加,S期和G2期细胞减少(P<0.05),增殖指数(40.4%)低于阴性对照(48.2%)(P<0.05)。Ed U增殖分析结果显示上调LincRNA-p21后,细胞增值率明显降低。Western blot结果显示过表达LincRNA-p21下调cyclin D蛋白水平。

[Objective] To investigate the underlying mechanisms of long intergenic non-coding RNA（LincRNA）-p21 on the inhibition of esophageal cancer cell proliferation.[Methods] The expression of LincRNA-p21 in two esophageal cancer cell lines（EC109 and EC9706）, a human immortalized esophageal epithelial cell line（Het-1A）, and the tissues and paired adjacent non-tumor mucosa of 64 cases of esophageal cancer were detected by real-time quantitative reverse-transcription polymerase chain reaction（RT-qPCR）. After being transfected successfully with lentivirus packaging full-length LincRNA-p21 gene, the cell cycle distribution of EC109 cells was detected by flow cytometry, and the proliferation of EC109 cells by Ed U staining. Meanwhile, RT-q PCR and Western blot were used to detect the mRNA and protein expression levels of p21 and the protein expression level of cyclin D.[Results] The LincRNA-p21 expression levels were 0.18 and 0.32 folds in EC109 and EC9706 cells as that in Het-1 A cells, respectively（P 0.05）, and the p21 mRNA levels were 0.42 and 0.48 folds, respectively. After transfection with lentivirus, the relative expression level of LincRNA-p21 increased by about 14 860 folds（P 0.05） and that of p21-mRNA increased by 1.83 folds（P 0.05）. The results of flow cytometry showed that in the LincRNA-p21 transfection group, cells in G1 phase increased, cells in S phase and G2 phase decreased（P 0.05）, and the proliferation index was lower than that in the negative control group（40.4% vs. 48.2%）（P 0.05）. The results of Ed U staining showed that the cell proliferation rate decreased after up-regulating LincRNA-p21. The results of Western blot showed that overexpression of LincRNA-p21 down-regulated the protein expression level of cyclin D.[Conclusion] LincRNA-p21 promotes the expression of p21 and inhibits the proliferation of human esophageal cancer EC109 cells by inhibiting the expression of cyclin D and contributing to G1/S arrest.